PROJECT SUMMARY/ABSTRACT The tumor microenvironment is known for its complexity, both in its content as well as its dynamic nature. It consists of various immune cells, stromal fibroblasts, extracellular matrix (ECM) components often associated with wound healing and inflammation, blood and lymph vessels, and various other cell types like endothelial progenitor cell. During cancer initiation and progression, these cells interact in a highly complex environment, which is not trivial to understand using two-dimensional models of tumor cell culture. Several advances in tissue engineering have allowed more physiologically-relevant in vitro tumor microenvironment models to be developed. Three-dimensional (3D) tumor spheroid cultures serve as powerful tools for dissecting the roles of various biochemical and biophysical cues in carcinoma initiation and progression. Although currently utilized microfluidic-based approaches enable cell-cell interactions in 3D, they lack the ability to control the organization of multiple cell types and cannot build complex 3D architectures, which typically requires manual manipulations of cells and biomaterials. The bioprinting approach proposed in this study will establish a biomimetic model platform that can be perfused through integration of built-in channels and recapitulates the cancer microenvironment. This model will advance our understanding of the fundamental interactions taking place between immune cells and cancer cells during cancer initiation and progression, and thereby reveal how these interactions can alleviate the progression of cancer. We hypothesize that cellular and molecular interactions between immune and tumor cells in 3D environments will differ from monolayer cultures and more closely recapitulate the in vivo state. To test this hypothesis, Specific Aim 1 will use bioprinting approaches to develop 3D models of breast cancer and human engineered T cells that can be activated by tumor cells within this in vitro microenvironment. In Specific Aim 2, we will determine the transcriptional impact of events within the 3D tumor- immune cell microenvironment compared to monolayer cultures through single cell RNA-sequencing approach. We will also assess the impact of different T cell subsets and myeloid lineage cells, such as macrophages and dendritic cells, on structural organization, angiogenic potential, metastatic behavior and cytotoxicity within the 3D tumor microenvironment. Further, we will test several approaches to enhance T cell responses to tumors in 3D bioprinted tissues, by CRISPR/Cas9 editing of antibody neutralization of T cell inhibitory molecules such as PD1 and CTLA-4. In this regard, we have formed a complementary collaboration that merges essential domain knowledge in human immunology, cell engineering and 3D bioprinting, with the depth necessary to propel the proposed work towards meaningful advances that would otherwise not be possible. Successful completion of the proposed work is anticipated to give rise to an integrated mechanistic framework revealing the complex interactions between immune cells and tumors in 3D biomimetic environment and thereby provide a novel understanding of how immune cells impact cancer intravasation and metastasis.